Unravelling the effects of hypoxia on purine biosynthesis in cancer

The purinosome is a dynamic metabolic complex composed of the six enzymes of the de novo purine biosynthesis that assembles in response to elevated purine demand. Its formation translates in an increase to the rate of the pathway in order to maintain the purine pool. This complex was first described to form in an artificially induced purine-depleted environment but later studies demonstrated its ability to form in pathological contexts, thus making the purinosome a promising therapeutic target to control purine synthesis. In various cancers, solid tumours often display hypoxic regions where oxygen deprivation induces a cellular response aiming at maintaining cell growth and proliferation, and contributes to the tumours resistance to treatments. In hypoxic cells, the heterodimeric transcription factor Hypoxia-Inducible Factor 1 (HIF-1) is responsible for the regulation of many target genes that ensure the response and adaptation of cancer cells to hypoxia. One of the most important adaptation mechanisms regulated by HIF is the metabolic reprogramming which supports the malignant phenotype of hypoxic tumours. As such, improving the current understanding of the metabolic adaptation to hypoxia is of high interest in order to further develop new therapeutic strategies to target hypoxic cancer cells. This work describes and investigates the formation of the purinosome complex in hypoxic cancer cells. Multiple cellular and biochemical approaches were used in order to characterise and understand the formation of the complex in low oxygen environments. The purinosome formation was found to be linked to HIF and to be modulated by various metabolic stimuli, thus indicating a link between the formation of the complex and its function. In contrast to its previously reported function in purine-depleted conditions, the hypoxic purinosome did not correlate with increased de novo synthesis of purines. This study lays the foundation for further investigations aiming at understanding the exact function of the purinosome in hypoxic cancer cells and raises the possibility that inhibiting purinosome formation in hypoxia might be of high therapeutic interest to specifically target hypoxic cancer cells

Optimized Protocol for Protein Macrocomplexes Stabilization Using the EDC, 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide, Zero-Length Cross-Linker

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Development of a cyclic peptide inhibitor of the p6/UEV protein- protein interaction

The budding of HIV from infected cells is driven by the protein-protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 μM by binding to UEV with a Kd of 11.9 ± 2.8 μM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of ∼2 μM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized

Hypoxia drives the assembly of the multi-enzyme purinosome complex

The purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis was observed in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand

Dataset for: Hypoxia drives the assembly of the multi-enzyme purinosome complex

Dataset supporting the paper: Doigneaux, C., Pedley, A., Mistry, I. N., Papayova, M., Benkovic, S., &amp; Tavassoli, A. (Accepted/In press). Hypoxia drives the assembly of the multi-enzyme purinosome complex. The Journal of Biological Chemistry. DOI:10.1074/jbc.RA119.012175</span

A lanthipeptide library used to identify a protein-protein interaction inhibitor

Dataset supports: Yang, X. et al. (2018). A lanthipeptide library used to identify a protein-protein interaction inhibitor. Nature Chemical Biology. This dataset contains raw data associated with Figures 3, 4 and the Supplementary Figures from the manuscript as detailed below: Figure 3a-i as Graphpad Prism files. Figure 4a as a Graphpad Prism file. Supplementary Figures 10, 11, 13, 15, and 16 as Graphpad Prism files. Sequencing data and script used to analyze data associated with Supplementary Figures 2, 3 and 9</span

Identification and development of cyclic peptide inhibitors of Hypoxia Inducible Factors 1 and 2 that disrupt hypoxia-response signaling in cancer cells

Hypoxia inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen- regulated α subunit and a constitutively expressed β subunit that serves as the master regulator of the cellular response to low oxygen concentrations. The HIF transcription factor senses and responds to hypoxia by significantly altering transcription, reprogramming cells to enable survival and growth in the hypoxic tumor microenvironment. Given the central role played by HIF in the survival and growth of tumors in a hypoxic microenvironment, inhibition of this transcription factor serves as a potential therapeutic approach for treating a variety of cancers. Here, we report the identification, optimisation and characterisation of a series of cyclic peptides that disrupt the function of HIF-1 and HIF-2 transcription factors by inhibiting the interaction of both HIF-1α and HIF-2α with HIF-1β. The resulting compounds are shown to bind HIF and disrupt the protein-protein interaction between the α and β subunit of the transcription factor, resulting in disruption of hypoxia-response signaling in several cancer cell lines

Dataset supporting the publication &quot;Identification and development of cyclic peptide inhibitors of Hypoxia Inducible Factors 1 and 2 that disrupt hypoxia-response signaling in cancer cells&quot;

Dataset supporting the publication: Ball, A. T., Mohammed, S., Doigneaux, C., Gardner, R. M., Easton, J. W., Turner, S., Essex, J. W., Pairaudeau, G., &amp; Tavassoli, A. (2024). Identification and development of cyclic peptide inhibitors of Hypoxia Inducible Factors 1 and 2 that disrupt hypoxia-response signalling in cancer cells. Journal of the American Chemical Society. The dataset includes raw data in Graphpad Prism format underpinning the publication: Figure 1d, Figure 2a, Figure 2b, Figure 3a, Figure 3d, Figure 4d, Figure 4e, Figure 5a, Figure 5b, Figure 5d, Figure 5e, Figure 5f, Figure S2, Figure S3b, Figure S3c, Figure S3d, Figure S4a, Figure S4b, Figure S4c, Figure S5a-o, Figure S6a-g, Figure S7a, Figure S7b, Figure S8 </span

A lanthipeptide library used to identify a protein-protein interaction inhibitor

We describe the production and screening of a genetically encoded library of 106 lanthipeptides in Escherichia coli using the substrate tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing significant structural diversity. Such libraries may be combined with any cell-based assay for the identification of lanthipeptides with new biological activities